1. Field of the Invention
The present invention relates to a biochemical reaction cassette having a probe carrier such as a DNA micro-array that can suitably be used as material for judging the health condition of a subject of examination by examining a specimen for the existence or non-existence of a gene originating from a pathogenic microbe in the specimen, which may typically be a blood specimen. More particularly, the present invention relates to the structure of a biochemical reaction cassette that is not expensive and shows an improved liquid filling performance.
2. Description of the Related Art
Techniques that utilize a hybridization reaction employing a probe carrier, which typically is a DNA micro-array, have been proposed for the purpose of quickly and accurately analyzing the base sequence of a nucleic acid or detecting the target nucleic acid in a nucleic acid specimen. A DNA micro-array is a set of nucleic acid fragments including a fragment having a complementary base sequence relative to that of the target nucleic acid, which fragments are referred to as probe and immobilized highly densely to a solid phase such as beads or a glass plate. The operation of detecting the target nucleic acid using a DNA micro-array generally has the steps as described below.
In the first step, the target nucleic acid is amplified by an amplifying method such as the PCR method. More specifically, the first and second primers are added into the nucleic acid specimen to begin with and a thermal cycle is applied to the specimen. The first primer specifically binds to part of the target nucleic acid while the second primer specifically binds to part of the nucleic acid that is complementary relative to the target nucleic acid. As double-stranded nucleic acids that include the target nucleic acid is combined with the first and second primers, the double-stranded nucleic acids including the target nucleic acid are amplified as a result of an extension reaction. As the double-stranded nucleic acids including the target nucleic acid are amplified sufficiently, the third primer is added to the nucleic acid specimen and a thermal cycle is applied to the specimen. The third primer is labeled with an enzyme, a fluorescent substance, a luminescent substance or the like and specifically combined with part of the nucleic acid that is complementary relative to the target nucleic acid. As the nucleic acid that is complementary relative to the target nucleic acid and the third primer are combined with each other, the target nucleic acid that is labeled with an enzyme, a fluorescent substance, a luminescent substance or the like is amplified as a result of an extension reaction. Then, consequently, the labeled target nucleic acid is produced when the nucleic acid specimen contains the target nucleic acid, whereas no labeled target nucleic acid is produced when the nucleic acid specimen does not contain the target nucleic acid.
In the second step, the nucleic acid specimen is brought into contact with a DNA micro-array to give rise to a hybridization reaction with the probe of the DNA micro-array. More specifically, the temperature of the DNA micro-array and the nucleic acid specimen is raised. Then, at this time, the probe and the target nucleic acid form a hybrid when the target nucleic acid is complementary relative to the probe.
In the third step, the target nucleic acid is detected. If, for instance, the labeling substance is a fluorescent one, the fluorescent substance is energized typically by means of a laser and the luminance of the energized substance is observed. In other words, it is possible to detect if the probe and the target nucleic acid has produced a hybrid or not by means of the labeling substance of the target nucleic acid and hence the presence or absence of a specific base sequence can be confirmed.
DNA micro-arrays adapted to utilize a hybridization reaction are expected to find applications in the field of medical diagnosis for identifying specific pathogenic microbes and gene diagnosis for examining bodily constitutions of patients. However, as a matter of fact, the step of amplification of the nucleic acid, that of hybridization and that of detection of the target nucleic acid as listed above are conducted normally individually by means of respective apparatus and involve cumbersome operations to make the diagnosis considerably time consuming. Particularly, when the hybridization reaction is made to take place on a glass slide, the probe can become missing or contaminated when the operator touches the glass slide with a fingertip because the probe-immobilizing region is exposed. Therefore, the operator is required to handle the probe very carefully. To avoid these and other problems, there have been proposed several biochemical reaction cassettes having a structure adapted to arrange a DNA micro-array in a reaction chamber, make a hybridization reaction to take place in the reaction chamber and conduct the subsequent detection step also in the reaction chamber.
FIGS. 7 and 8 illustrate such a biochemical reaction cassette. FIG. 8 is a cross sectional view of the biochemical reaction cassette of FIG. 7 taken along a plane parallel to the vertical direction that includes the injection port and the discharge port. Referring to FIGS. 7 and 8, the biochemical reaction cassette 51 comprises a housing 52 and a glass substrate 53 to which a DNA probe that is to specifically bind to a target nucleic acid is immobilized. The housing 52 is provided with a dent section (recess) and part of the recess forms a reaction chamber 54 having a bottom surface where the DNA probe is immobilized as the housing 52 and the glass substrate 53 are bonded to each other. An injection flow channel 55 and a discharge flow channel 56 are connected to the reaction chamber 54 so that the liquid specimen to be analyzed and one or more than one reagents may be injected and discharged.
The reaction chamber 54 of the biochemical reaction cassette 51 as illustrated in FIGS. 7 and 8 has only a small volume of tens of several microliters and bubbles are apt to remain in the reaction chamber 54 after filling it with liquid due to its structure. The biochemical reaction can be blocked and the diagnosis can be adversely affected when bubbles remain in the region where the DNA probe is immobilized to the glass substrate 53. The operation of precisely controlling the movement of liquid so that bubble may not remain in the reaction chamber 54 is a cumbersome one and additionally such bubbles can form an obstacle when the biochemical reaction cassette is applied to an automatic diagnostic apparatus. To avoid this problem, Japanese Patent Application Laid-Open No. 2003-302399 discloses an arrangement where the reaction chamber is provided on the upper or lower surface thereof with a hydrophobic region and a hydrophilic region. Japanese Patent Application Laid-Open No. 2004-093558 discloses an arrangement for preventing bubbles from being produced by means of a flow channel formed by using a protruding member in an upper part of the reaction region. Japanese Patent Application Laid-Open No. 2002-243748 discloses an arrangement for forming a uniformly spreading flow of liquid by means of a butterfly structure or a cascade structure.
The arrangement of Japanese Patent Application Laid-Open No. 2003-302399 and that of Japanese Patent Application Laid-Open No. 2004-093558, however, cannot completely eliminate bubbles remaining at and near the outlet port. Similarly, with the arrangement of Japanese Patent Application Laid-Open No. 2002-243748, bubbles may be left in an upper part of the reaction chamber because the outlet port is connected to an end of the chamber. When bubbles are left at and near the outlet port, they can grow in the hybridization step to cover the DNA probe-immobilizing region because of the temperature rise in that step. Then, the biochemical reaction can be blocked to adversely affect the diagnosis.
Additionally, the arrangements of Japanese Patent Application Laid-Open No. 2003-302399, Japanese Patent Application Laid-Open No. 2004-093558 and Japanese Patent Application Laid-Open No. 2002-243748 require the cassette to be surface-treated and involve a complex profile for the reaction chamber to consequently raise the cost of manufacturing the cassettes.